Bypassing mutant p53 in radiation therapy

在放射治疗中绕过突变型 p53

• 批准号: 8867861
• 负责人: Hongdau Peter Liu
• 金额: $ 4.81万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2017-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8867861
• 关键词: Aftercare; Apoptosis; Apoptotic; Biological Markers; Blinded; Bypass; Cancer Patient; Cancerous; Cell Culture Techniques; Cell Cycle Arrest; Cell Death; Cells; Cleaved cell; Clinical; DNA; DNA Damage; Embryo; FDA approved; Fishes; Genetic; Genetic Determinism; Genetic study; Hela Cells; Human; Ionizing radiation; Libraries; Malignant Neoplasms; Mediating; Methods; Modeling; Mutate; Mutation; PTEN gene; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Preclinical Drug Evaluation; Process; Radiation; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radioresistance; Radiosensitization; Regulation; Research; Resistance; Signal Transduction; Specificity; Stratification; TP53 gene; Testing; Therapeutic; Tumor Suppressor Proteins; Zebrafish; base; cancer cell; cancer therapy; caspase-2; chemical genetics; chemotherapy; clinically relevant; improved; in vivo; inhibitor/antagonist; irradiation; mutant; neoplastic cell; novel; novel therapeutic intervention; oncology; protein complex; public health relevance; radiosensitizing; resistance mechanism; response; screening; small molecule; small molecule libraries; therapeutic target; tumor

Project Summary Mutation of the tumor suppressor TP53 leads to the evasion of apoptosis after radiation, underlying resistance to conventional cancer treatments, such as radiation and chemotherapy. Our research seeks to bypass these alterations through the discovery of parallel cell-death pathways. We previously discovered a novel apoptotic process, termed "Chk1-suppressed" (CS) pathway, which restores radiosensitivity in TP53 mutant cells after treatment with Chk1 inhibitors (Sidi et al. Cell 2008)1. Using both zebrafish and mammalian TP53-mutant models, our lab has established that the CS pathway requires the assembly of a protein complex called the PIDDosome, composed of PIDD, RAIDD, and caspase-2 (Ando et al. Mol Cell 2012)2. This proposal focuses on (1) identifying further genetic determinants predicting Chk1 inhibitor efficacy in radiotherapy and (2) discovering novel radiosensitizers against tumors with TP53 mutation. Utilizing both human cancer cells and zebrafish, we investigated the impact of cancer alterations other than mutant p53 on cellular sensitivity to CS apoptosis. We found that a mutation in PTEN, the second most commonly mutated tumor suppressor, blocks the execution of apoptosis after ionizing radiation (IR) and Chk1 inhibition (Chk1i). Furthermore, we identified the PTEN-Akt-IAP signaling axis as a candidate pathway to promote resistance to CS apoptosis. The loss of PTEN appears to inhibit the CS pathway downstream of caspase-2 activation, as HeLa cells with impaired PTEN fail to engage in apoptosis, despite an unaffected ability to cleave caspase-2 following IR+Chk1i. Our hypothesis is that one or more IAPs act downstream of Akt to directly inhibit active caspase-2. I propose to test this hypothesis using a candidate knockdown approach to determine which IAP(s), if any, is responsible for caspase-2 inhibition. The findings will be extended in vivo through both chemical and genetic studies in zebrafish. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput drug screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. A primary screen of drugs treated in combination with 15 Gy IR yielded 140 compounds capable of producing phenotypes comparable to Chk1i (i.e. >75% sick or dead fish 4 days post irradiation) in p53 mutant fish. Our secondary screen of irradiated vs non-irradiated embryos so far has identified 6 compounds that demonstrate specific radiosensitizing effects. The genetic specificity of these candidate radiosensitizers will be confirmed and prioritized by clinical relevance, proposed mechanism of action, and efficacy. The proposed targets, along with the mechanism(s) of cell death will be confirmed in cell culture and in vivo. The results of these findings may aid in rapidly benefiting cancer patients by proposing a new application for an already approved and available drug.

项目摘要 肿瘤抑制因子TP 53的突变导致辐射后细胞凋亡的逃避， 对常规癌症治疗如放疗和化疗的抵抗。我们的研究旨在 通过发现平行的细胞死亡途径来绕过这些改变。我们之前发现了一个 一种新的凋亡过程，称为“Chk 1抑制”（CS）途径，可恢复TP 53的放射敏感性 用Chk 1抑制剂处理后的突变体细胞（Sidi等人，Cell 2008）1.利用斑马鱼和哺乳动物 TP 53突变模型，我们的实验室已经确定，CS途径需要组装蛋白质复合物 称为PIDDosome，由PIDD、RAIDD和半胱天冬酶-2组成（Ando et al. Mol Cell 2012）2.这项建议 重点是（1）确定进一步的遗传决定因素，预测Chk 1抑制剂在放射治疗中的疗效，以及（2） 发现针对具有TP 53突变的肿瘤的新型放射增敏剂。 利用人类癌细胞和斑马鱼，我们研究了癌症改变的影响， 突变型p53对CS凋亡敏感性的影响。我们发现，在第二大基因组中， 通常突变的肿瘤抑制因子，阻断电离辐射（IR）和Chk 1后细胞凋亡的执行 抑制（Chk 1 i）。此外，我们确定了PTEN-Akt-IAP信号传导轴作为一个候选途径， 促进CS凋亡的抵抗。PTEN的缺失似乎抑制了CS途径下游的 caspase-2激活，因为PTEN受损的HeLa细胞不能参与凋亡，尽管未受影响， IR+ Chk 1 i后切割caspase-2的能力。我们的假设是一个或多个IAP在Akt下游起作用 直接抑制活性半胱天冬酶-2。我建议使用候选击倒方法来测试这一假设， 确定哪种IAP（如果有的话）负责caspase-2抑制。研究结果将在体内进行扩展 通过对斑马鱼的化学和遗传研究。 为了确定恢复TP 53突变肿瘤放射敏感性的新靶向策略，我们利用 斑马鱼进行盲法表型放射增敏的高通量药物筛选能力 筛选了640种FDA批准的化合物。15戈伊放疗联合用药的初步筛选 产生了140种能够产生与Chk 1 i相当的表型的化合物（即>75%的病鱼或死鱼4 照射后30天）。到目前为止，我们对辐照与非辐照胚胎的二次筛选 已经确定了6种化合物，表现出特定的放射增敏作用。这些基因的特异性 候选放射增敏剂将通过临床相关性、拟定的 作用和功效。将在细胞中证实所提出的靶点，沿着细胞死亡的机制。 培养和体内。这些发现的结果可能有助于通过提出一种新的治疗方法来迅速使癌症患者受益。 已批准和可用药物的新申请。